H1 linker histone is a major structural component of chromatin being nearly equimolar with nucleosome core particles. The interaction of histone H1 with nucleosome core particles and linker DNA promotes the folding of the chromatin fiber into a compact structure, and, therefore, is likely to affect most transactions occurring on DNA, including regulation of gene expression. Mammals have eight histone H1 subtypes. By generating histone H1 knockout mice, our laboratory showed that these subtypes can substitute for one another in fundamental aspects of chromatin function. However, reducing the stoichiometry of total histone H1 proteins in chromatin by generating triple H1 null mice results in midgestational embryonic lethality due to a wide variety of developmental defects. This embryonic lethality has prevented attempts to study the role of H1 in later development. To determine the in vivo functions of histone H1 in later (fetal and adult) mouse development, conditional triple HI null mice will be generated using the Cre-LoxP system. Using Cre- recombinase expressed specifically in liver we will determine the effects of depletion of histone H1 on liver development and liver-specific gene expression. To complement these studies, RNA interference will be used to reduce histone H1 levels further in the triple H1 null ES cells already available in our laboratory. Previous work showed that the triple H1 null ES cells, having about 50% the wild-type level of histone H1, proliferate normally and have changes in expression of a small number of genes. We will determine whether a further reduction (below 50%) of H1 levels in ES cells results in additional, specific gene expression changes, as well as changes in cell proliferation, survival, mitosis and replication. These experiments will provide insight into the in vivo functions of histone H1 proteins, and will further our knowledge about chromatin organization. Elucidating the role of chromatin structure and chromatin proteins in gene expression will increase our knowledge about the role of chromatin in normal mammalian development and disease processes. Histone H1 is a protein involved in packaging immense amounts of DNA into cells. Proper packaging of DNA is essential for correct turning on and off of genes; incorrect packaging can cause many problems in cells, leading to diseases, such as cancer. We are studying histone H1 in mice to understand both how histone H1 helps to ensure the proper control of the genes and the role it plays in mouse development, a complex organism sharing many developmental features with humans. [unreadable] [unreadable] [unreadable]